DE3744595A1 - METHOD FOR THE GENETIC ENGINEERING OF ANTIBODY - Google Patents

Abstract

Functional antibody fragments can be produced in bacteria by coupling each of the genes for the individual chains to signal sequences which bring about the transport of the prepeptides through the cytoplasmic membrane, expressing the gene structures in a bacterium, and isolating the functional protein from the periplasmic space or the medium. The functional protein is advantageously isolated by affinity chromatography on an adsorbent which is loaded with the hapten or antigen, and elution with a solution of the hapten or antigen.

Description

The expression of antibodies in yeast has been described (C.R. Wood, M.A. Boss, J.H. Kenten, J.E. Calvert, N.A. Roberts and J. S. Emtage, Nature 314, 446 (1985)), but only a very small fraction of the expressed protein turned out to be functional. So far in E. coli Antibody proteins only obtained in denatured form ((M.A. Boss, J.H. Kenten, C.R. Wood and J.S. Emtage, Nucleic Acids Res. 12, 3791 (1984); S. Cabilly, A.D. Riggs, H. Pande, J.E. Shively, W.E. Holmes, M. Rey, L.J. Perry, R. Wetzel and H.L. Heyneker, Proc. Natl. Acad. Sci. UNITED STATES. 81: 3273 (1984)). The cleaning of active antibodies or Antibody fragments from yeast or other microorganisms it is unknown yet. Protein folding attempts have so far only a small percentage of correctly folded recombinant antibody proteins. Besides, it is difficult to get the desired functional proteins from to separate unwanted and non-functional proteins, what the exact measurement of binding constants, yields at convolution, spectral properties and the like and the application in therapy and technology difficult. The Optimization of the folding conditions is therefore extremely difficult, especially since there are no proven ones Procedures and measuring methods for refolding there.

The expression of functional complete antibodies or functional binding domains of antibodies in bacterial expression systems are not yet known and the prospects have become real pessimistic (S. L. Morrison, Science 229, 1202 (1985), M.A. Boss and C.R. Wood, Immunol. Today 6, 12 (1985)).

Such a system would be very desirable since the genetic engineering processes particularly good for E. coli  are developed and due to the rapid growth the Mass production is easier from what is economically is of considerable importance.

The invention therefore relates to the production of functional antibodies, their functional fragments or fusion proteins from antibody domains and others Proteins in bacteria, preferably gram-negative Bacteria, especially in E. coli. The invention The method is characterized in that the genes for the individual chains of the antibody molecule or fragment each coupled to a signal sequence that the transport of the Polypeptide chains caused by the cytoplasmic membrane and can be split off, the gene structures for Expression brings and the functional protein from the periplasmic space or the medium isolated. Preferred Embodiments of this invention are described in more detail below explained or defined in the claims.

The coupling of genes for the individual with signal Sequenced chains are advantageously of the type of an adjustable operon system that allows simultaneous Expression caused by a common control region. In this way, the individual protein chains in the approximately stoichiometric ratio expressed together and transported into the periplasmic space, where the Connection to the functional molecule is made. The transport of proteins from the cytoplasm occurs per se known methods, such as those in the EP-B 00 06 694 are described.

Any suitable controllable region comes as a control region Gene regulation region into consideration, for example lac, tac trp or synthetic sequences. Are particularly preferred Regulatory regions that can be reliably switched off.

Isolation of proteins from the periplasmic space  is advantageously done by looking at the harvested Exerts a mild osmotic shock and the liquid phase obtained by ultrafiltration or precipitation, for example with salts such as ammonium sulfate, concentrated.

A "mild" osmotic shock causes the Periplasmas, but with the cytoplasmic membrane intact remains.

The protein concentrate is useful after dialysis on an adsorbent, advantageously in the form of a Affinity column, given that with the corresponding Is loaded with antigen or hapten. By suitable elution, advantageous with the antigen or hapten, you then get the Antibodies or the functional antibody fragment.

In the eukaryotic cell, antibodies in the lumen of the endoplasmic reticulum - probably under Contribution of disulfide isomerases, proline-cis-trans Isomerases and possibly other enzymes or Proteins - formed. It was surprising that the Bacterial cell is able to link the two chains in the cell to produce about the same stoichiometric amount, both Precursor proteins in the periplasmic space or they surrounding medium to transport the signal sequences correctly split off the globular and soluble domains to fold correctly, the intramolecular disulfide formation form and associate both chains into a heterodimer, since it is considered unlikely that the bacterial cell over has such or equivalent enzymes. It was found So surprisingly, in the case of grief-negative Bacteria the bacterial periplasm the lumen of the eukaryotic endoplasmic reticulum in this Relationship is functionally equivalent.

The method according to the invention has a number of advantages:  

Apart from the light and cheap already mentioned bacterial mass fermentation become immediate functional proteins formed, thus causing the cleavage of fusion proteins with subsequent isolation of the desired protein or protein fragment, its Oxidation or in vitro refolding can be avoided.

There were also no problems with the method according to the invention detected with cellular proteases. As you know, yes because of these cellular proteases proteins in bacteria usually in the form of fusion proteins, especially as an insoluble inclusion body, which expresses what mentioned elaborate processing steps with it brings. In contrast, the separation and cleaning up to Homogeneity in the method according to the invention quickly and easy.

The invention thus allows easy access to Antibodies, their functional fragments and modified antibodies, which are characterized by insertion, Elimination and / or replacement of amino acids from the distinguish native antibodies. So you can for example eliminating cysteine or by others Replace amino acids to prevent unwanted folding suppress. You can also use a murine antibody in convince a human, or introduce other mutations. In addition to the pharmacological and technical Applications are therefore easier Access to research into antibody structure and function and the basics of enzymatic catalysis (V. Raso and B. D. Stollar, Biochemistry 14: 584 (1975); V. Raso and B. D. Stollar, Biochemistry 14: 591 (1975); A. Tramontano, K.D. Janda and R.A. Lerner, Science 234, 1566 (1986); S. J. Pollack, J.W. Jacobs and P.G. Schultz, Science 234, 1570 (1986); J. Jacobs, P.G. Schultz, R. Sugasawara and M. Powell, J. Am. Chem. Soc. 109, 2174 (1987).  

The invention also allows the use of Test systems (assays) directly on the bacterial cell, in which the functional antibody is formed, and thus a quick check for mutations if necessary Antibody.

In addition to the above-mentioned variations of the antibody molecule Variation of single or fewer amino acids can be found in the Antibody gene also inserted other gene regions or parts be exchanged for non-critical gene regions. One can so labeling enzymes (M. S. Neuberger, G. T. Williams and R. O. Fox, Nature 312, 604 (1984)), toxins (G. Möller ed.) "Antibody carriers of drugs and toxins in tumor therapy", Immunol. Rev. 62, Munksgaard, Copenhagen (1982)) or Another class of immunoglobulin regions (M. S. Neuberger, G. T. Williams, E. B. Mitchell, S. S. Jouhal, J. G. Flanagan, and T. H. Rabbitts, Nature 314, 268 (1985)) or one other species (P. T. Jones, P. H. Dear, J. Foote, M. S. Neuberger, and G. Winter, Nature 321, 522 (1986)) to the Coupling the antibody molecule.

The method according to the invention is explained below using the example of the variable domains of the phosphorylcholine-binding antibody-myeloma protein McPC 603. The three-dimensional structure of this mouse immunoglobulin A is known (DM Segal, EA Padlan, GH Cohen, S. Rudikoff, M. Potter and DR Davies, Proc. Natl. Acad. Sci. 71, 4298 (1974)). Synthetic genes were used for the variable light chain V _L and heavy chain V _H. Such synthetic genes are proposed in German Patent Application P 37 15 033.2 (Tables 1 and 2 there). A particularly expedient embodiment of such synthetic genes is shown in the table, in which the DNA sequence of the entire expression plasmid is shown and the genes for the two chains are highlighted by the amino acid details. Codons that are rarely used by E. coli were avoided in the construction of these DNA sequences. Furthermore, unique restriction enzyme interfaces were built in and the secondary structure of the RNA was taken into account.

In the functional antibody fragment serving as a model, each domain has an intramolecular disulfide bridge (from Cys 23 to Cys 94 in V _L and from Cys 22 to Cys 98 in V _H ). There is no disulfide bridge between the chains and no free cysteine.

The expression vector used is shown schematically in FIG . 1 reproduced. In it, the synthetic genes for the V _L and V _H domains are linked to gene fragments for the bacterial signal sequence of the outer membrane protein A (ompA) on the one hand and the alkaline phosphatase (phoA) on the other. The genes for the two precursor proteins are arranged in an artificial operon-like structure behind the lac promoter, which ensures the simultaneous induction, co-expression and co-secretion of both genes.

After induction of gene expression, the cells are harvested and subjected to a mild osmotic shock. The liquid phase obtained in this way, which contains the periplasmic proteins, is concentrated by ultrafiltration, dialyzed and applied directly to an affinity column which contains a phosphorylcholine derivative (B. Chesebro and H. Metzger, Biochemistry 11, 766 (1972)) as affinity ligands. Elution with phosphorylcholine gives a homogeneous F _V fragment which is uniform in gel electrophoresis. It can be deduced from the SDS polyacrylamide gel that both chains of the purified F _V fragment are present in a molar ratio of 1: 1 and that the expected molecular weights of the mature proteins (V _H : 13 600 D, V _L : 12 400 D) are present . The six N-terminal amino acids of both chains were sequenced to demonstrate the correct cleavage of both signal sequences. It was found that both chains had the correct N-termini for the mature proteins. Both heterologous preproteins were therefore correctly cleaved by the bacterial signal peptidase and there is no evidence that an imprecise processing or an N-terminal degradation reaction occurred.

The affinity constant of the McPC603 recombinant F _V fragment was measured by equilibrium dialysis. The same conditions were used here, which were used in the determination of the affinity constant of the native antibody McPC603, isolated from mouse ascites. The value of 1.21 ± 0.06 × 10 ^-5 l / mol found for the F _V fragment is identical within the experimental accuracy to the value 1.6 ± 0.4 × 10⁵ l / mol reported for the native antibody. The Scatchard binding curve (Ann. NY Acad. Sci. 51, 660 (1949)) is linear and the extrapolation indicates that approximately 1 mole of hapten is bound per mole of F _V fragment. This confirms that there is only one type of binding site per F _V fragment and that there are no inactive components in the isolated protein.

It has surprisingly been found that the F _V fragment of the McPC603 antibody can be produced as a completely functional and stable protein in E. coli. This proves the functional equivalence of the transport into the periplasm of the bacterial cell for transport into the lumen of the endoplasmic reticulum of the eukaryotic cell. This equivalence is unknown and also unexpected, since the best characterized bacterial protein to date, which could be defined as soluble heterodimeric protein in the periplasm, E. coli penicillin acylase, is produced by proteolytic processing from a single-chain precursor in the periplasm (G. Schumacher, D. Sizmann, H. Hang, P. Buckel and A. Böck, Nucleic Acids Res. 14, 5713 (1986)). Furthermore, it has already been pointed out that, according to current knowledge, bacteria do not have any enzymes or proteins that could be involved in folding in eukaryotes.

It was also surprising that the McPC603 F _V fragment has essentially the same affinity constant for phosphorylcholine as the intact McPC603 antibody itself. This finding is unexpected, since the functionality of F _V fragments is controversial in the literature (J. Sen and S. Beychok, Proteins 1, 256 (1986)). This shows that a modification or even complete deletion of the constant domains can maintain the functionality.

In contrast to the only method for the production of F _V fragments so far, namely by proteolysis of an antibody, the method according to the invention has no problems with non-functional, incorrectly folded, incorrectly reassociated or chemically modified proteins. Moreover, the preferred isolation method with the aid of an adsorbent loaded with antigen or hapten is also suitable for purifying such F _V fragments obtained by known methods, since such impurities would either not be bound or would not be eluted.

example 1 Preparation of the plasmid pASK22

The expression plasmid pASK22 is the major EcoRI-HindIII fragment from pUC12 (C. Yanisch-Perron, J. Vieira, and J. Messing, Gene 33, 103 (1985)), from fragments of the vectors pIN III-OmpA1 (Y. Masui, J. Coleman and M. Inouye in "Experimental manipulation of gene expression", M. Inouye, ed., Academic Press 1983) and pHI61 (H. Inouye, W. Barnes and J. Beckwith, J. Bacteriol. 149, 434 (1982)) and different synthetic DNA fragments in several Stages using methods known per se (T.  Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning, Cold Spring Harbor 1982). The complete DNA Sequence of the vector pASK22 thus obtained is in the table shown.

With the ligation mixture competent E. coli cells are transformed and selected for ampicillin resistance. The Plasmids with the desired gene structure are identified by Restriction analysis and sequencing of the critical Characterized transitions.

Example 2 Production of the F _V fragment

A culture of the E. coli strain W3110 transformed with pASK22 is grown in LB medium with 100 mg / l ampicillin to an OD₅₅₀ of 0.5. Expression is induced by adding IPTG up to a final concentration of 1mM. After 45 minutes, the cells are harvested by centrifugation at 4000 g (10 minutes at 4 ° C). A cell fractionation is carried out by resuspending the cell pellet in TES buffer (0.2 M Tris.HCl, pH 8.0; 0.5 mM EDTA; 0.5 M sucrose) in 10 ml / l of the original culture. The cells are subjected to mild osmotic shock by adding 15 ml / l of the original culture TES buffer, which is diluted 1: 4 with water and contains 2 mM phosphorylcholine. After 30 minutes of incubation on ice, the suspension is centrifuged at 5000 g for 10 minutes and the supernatant is centrifuged again at 48,000 g for 15 minutes. The supernatant thus obtained, which contains all soluble periplasmic proteins, is concentrated by ultrafiltration (®AMICON YM5 membrane) to a volume of about 2.5 ml / l of the original culture and against BBS buffer (0.2 M borate / NaOH, pH 8.0; 0.16 M NaCl) dialyzed. This concentrated solution is placed on an affinity column loaded with a phosphorylcholine derivative (B. Chesebro and H. Metzger, loc. Cit.) (1-4 ml solution per ml bed volume), washed with BBS buffer and the pure F _V fragment eluted with a solution of 1 mM phosphorylcholine in BBS buffer.

Example 3 Balance dialysis

In a dialysis chamber with a volume of 100 µl on each side of the membrane, 50 µl of purified F _V fragment in BBS buffer were placed on one side and a solution of 50 µl phosphoryl (methyl-1 C) choline (50 mCi / mol ) filled in BBS buffer. From the OD₂₀₅ of 6.85, a concentration of 0.22 mg / ml was determined for the F _V fragment (RK Scopes, Protein Purification Principles and Practice, Springer-Verlag, New York, 1982, p. 241). After an equilibrium of 22 hours at room temperature, 20 ul samples of each solution were measured in a scintillation counter (Beckman LS 1801) and the data according to Scatchard (op. Cit.) Were plotted. The slope of the line obtained gives an affinity constant of K _a = 1.21 ± 0.06 × 10⁵ M ^-1 .

table

DNA sequence from pASK22 with amino acid sequences from ompA-V _H and phoA-V _L

Claims (6)

1. A process for the production of functional antibodies, functional fragments of antibodies or fusion proteins from antibody domains and other proteins, characterized in that the genes for the individual chains are each coupled to a signal sequence which bring about the transport of the polypeptide chains through the cytoplasmic membrane of a bacterial cell and then cleaved off, expressing the gene structures in a bacterium and isolating the functional protein from the periplasmic space or the medium. 2. The method according to claim 1, characterized in that the bacterium is a gram-negative bacterium. 3. The method according to claim 2, characterized in that the bacterium is E. coli. 4. The method according to claim 1, 2 or 3, characterized characterized in that the genes for the two prepeptides couples in the form of an adjustable operon system. 5. Method according to one or more of the preceding Claims, characterized in that for insulation of the functional protein from the periplasmic space the separated bacteria from such an osmotic shock exposes that the periplasm emerges, but the cytoplasm remains intact, the resulting liquid phase through Centrifuging enriches and from this concentrate the desired protein wins. 6. Method according to one or more of the preceding Claims, characterized in that the functional protein containing solution on a with the corresponding antigen or hapten loaded adsorbent and the desired proteins by elution with one of these Solution containing antigen or hapten eluted.